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Virions from hog cholera virus (HCV), a member of the genus Pestivirus, were analyzed by using specific antibodies. The nucleocapsid protein was found to be a 14-kDa molecule (HCV p14). An equivalent protein could also be demonstrated for virions from another pestivirus, bovine viral diarrhea virus. The HCV envelope is composed of three glycoproteins, HCV gp44/48, gp33, and gp55. All three exist in the form of disulfide-linked dimers in virus-infected cells and in virions; HCV gp44/48 and gp55 each form homodimers, whereas gp55 is also found dimerized with gp33. Such complex covalent interactions between structural glycoproteins have not been described so far for any RNA virus.

Negatively stained preparations of rotavirus imaged with a low dose of electrons provide sufficient contrast to reveal surface projections or spikes. The number of spikes found projecting from different particles indicates that not all 60 peripentonal sites are occupied. Treatment at pH 11.2 with 250 mM ammonium hydroxide specifically removes the spikes, yielding smooth double-shelled particles of the same diameter as that of the native virus. Protein analysis confirms that the released spikes are composed of polypeptide VP4 (or its two cleavage products VP5* and VP8*) and that the smooth particle retains the other major outer shell protein VP7. Spikeless particles can be decorated by a monoclonal antibody specific for the major immunodominant neutralizing domain of VP7, implying that removal of the spikes does not denature the VP7 that is retained on the surface of the smooth particle.

Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.

Both the supernatant of influenza virus-infected chick embryo cells and allantoic fluid containing influenza virus were shown to contain non-virion nucleoprotein (NP), which reacted readily with anti-NP monoclonal antibodies. Adsorption onto erythrocytes and centrifugation at 70,000 g for 2 h resulted in the removal of about 20% of the extracellular NP, whereas centrifugation at 100,000 g for 4 h eliminated about 50%, and practically all [3H]uridine-labelled virions. These results suggest that of the extracellular NP about 30% exists in the form of ribonucleoprotein, about 20% is precipitated with virions and about 50% occurs as free molecules. Comparative analysis of the kinetics of the accumulation of NP in the supernatant of infected cells, on the cell surface and inside the cells in relation to virus production, showed that there is a significant correlation between them.

Polyclonal antisera raised against fusion proteins containing portions of the vaccinia virus D2L and D3R proteins were prepared. Immunoprecipitation of pulse-labeled infected cell extracts and Western blot analysis demonstrated that genes D2L and D3R encode 16.9- and 27-kDa proteins, respectively. Both are synthesized late during infection and there is no evidence for proteolytic processing of either protein. Western blots of purified virus and subvirion fractions showed that D2L and D3R are virion components, residing in a detergent-insoluble fraction, containing viral core structural proteins. Trypsin sensitivity experiments suggest that each is found in an equivalent position within the virus core. Pulse-chase analysis showed that both proteins exhibit biphasic stability in which an unstable nascent component is replaced by a stable form. This observation suggests that the stable component results from the insertion of D2L and D3R into an immature core structure. The DNA sequence of four ts mutants previously mapped to genes D2L and D3R is reported. Analysis of the ability of each mutant to synthesize and process viral proteins showed that protein synthetic patterns were indistinguishable from wild type, however, three of the four mutants were defective in the processing of the major virion structural precursor, p4a. Unlike the biphasic stability observed in wild-type infected cells, D2L and D3R were totally degraded in cells infected at 40 degrees with any of the four ts mutants. Stability of the D2L and D3R proteins, in cells treated with rifampicin, is unaffected which demonstrates that a block in morphogenesis is not directly responsible for the observed instability of the mutant proteins.

A complete cDNA copy of the fifth RNA segment of the human group B rotavirus, ADRV, has been cloned into plasmid AD512. Gene segment 5 contains 1269 bases and encodes one long open reading frame of 391 amino acids beginning at base 31 and terminating at base 1203. The gene 5 polypeptide, expressed in vitro in a rabbit reticulocyte lysate, comigrates with the 44-kDa major inner capsid protein present on EDTA treated ADRV virions. The gene 5 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent serum and by a group B-specific monoclonal antibody. In addition, this protein shares amino acid identity and similarity with the VP6 proteins from group C and group A rotavirus strains. The ADRV VP6 equivalent protein appears to be more closely related to the group C VP6 than the Group A VP6 polypeptide and a common ancestral rotavirus VP6 precursor protein is suggested. As a result, the fifth RNA segment of ADRV defines the major inner capsid protein, or VP6 equivalent, in the group B rotavirus. Expression of the ADRV VP6 equivalent protein is potentially useful for evaluating the prevalence of serum antibodies to group B rotavirus in human and animal populations as well as for generating antibodies for the direct detection of group B rotavirus antigen.

A cytoplasmic dsRNA virus, rotifer birnavirus (RBV), has recently been isolated from the rotifer Brachionus plicatilis and is associated with a high mortality rate. Histologically, the viral lesions consist of characteristic inclusions, particularly amorphous dense bodies containing occluded particles. Purified virions are about 59 nm in diameter, single-shelled and display four capsomers per edge. The purified virions have a buoyant density of 1.290 (full particles) and 1.250 (empty particles) in CsCl gradients. Four major structural polypeptides of MrS 60K, 52K, 33K and 27K were detected by SDS-PAGE. The genome is composed of two linear segments of dsRNA with MrS of 2.45 x 10(6) and 2.31 x 10(6); additionally, small circular ssRNA molecules were detected by electrophoresis in overloaded agarose gels, but their significance is currently unknown. Except for this last feature and the structural instability of purified virions under freeze storage, all the other biochemical and biophysical characters indicate that RBV is a member of the Birnaviridae family with, for the moment, a unique position in this group.

The isoelectric point (pI) values of matrix protein M1 of influenza A, B, and C viruses, calculated theoretically on the basis of its primary structure, were found to be about 10.0. However, experimental pI determination by electrofocusing in ampholyte-containing polyacrylamide gel showed it to be 5.0 for M1 protein isolated from A/WSN/33 (H1N1) and A/Aichi/2/68 (H3N2) viruses by mild deproteinization with nonionic detergents. This marked discrepancy between experimental and theoretical pI values indicated that influenza virus matrix protein M1 possessed an unusual tertiary structure and/or intensive posttranslational addition of charged residues.

Capsid protein VP4 of poliovirus is acylated with myristic acid via an amide linkage to its N-terminal glycine residue. Our previous studies suggested that myristic acid plays a role in poliovirus assembly and in the early events of infection. In order to understand better its role in the assembly process, we introduced a Gly1 to Ala amino acid substitution in the myristoylation signal sequence of VP4. This substitution prevented VP0 myristoylation in vivo and abolished the infectivity of genomic transcripts harbouring the mutation. These mutated RNAs were still able to replicate in the transfected cells but the assembly processes were inefficient and no mature virions could be detected.

The present paper reports two new formulas for calculating triangulation number T. T = L2/l2 and T = 1.45 r2/l2, where L is the distance between pentons, l the distance between any two adjacent capsomeres, r the radius of viral nucleocapsid. The formulas have been verified and applied. It is worth noticing that the triangulation number, viral size and distance between capsomeres are fully connected by the formula r/the square root of Tl = 0.83, and the capsid parameters of all icosahedral viruses are unified in one constant, 0.83.

The 3'-terminal region of wheat streak mosaic virus (WSMV) genomic RNA was cloned and a cDNA sequence of 1809 nucleotides upstream of the poly(A) tract was determined. The sequence contains a single open reading frame of 1662 nucleotides and a 3' untranslated region of 147 nucleotides. Translation products from WSMV RNA and WSMV cDNA transcripts were immunoprecipitated by WSMV capsid protein antiserum, indicating that the 3'-terminal region of WSMV RNA encodes the capsid protein. Five potential N-terminal capsid protein protease cleavage sites were identified, which would yield proteins ranging from 31.7K to 46.8K. Alignment of the deduced amino acid sequence of the WSMV capsid protein with those of other potyviruses showed significant, but limited, identity as compared to the alignment of two or more aphid-transmitted potyviruses. Although WSMV has characteristics distinct from potyviruses, because of its particle morphology, translation strategy apparently based on polyprotein processing, the ability to form cytoplasmic cylindrical inclusions and the degree of capsid protein homology with aphid-transmitted potyviruses, it should be considered a member of the potyvirus group.

During gradient purification of herpes simplex virus type 1 (HSV-1) two bands of particles were observed: a sharp lower band and a more diffuse upper band. The lower band contained almost exclusively HSV-1 virions (H particles) whereas the upper band consisted of membrane-enclosed particles (L particles). These L particles resembled the virions in appearance, but lacked the viral nucleocapsid and were not infectious. Many polypeptides of the viral envelope and the tegument were common to both types of particles. The H particles had polypeptide profiles typical of HSV virions. The L particles contained at least three phosphoproteins (175K, 92K and 55K) and a further two phosphorylated polypeptides not normally observed in virion profiles which comigrated with the 134K and 60K glycoproteins. This clearly indicates that the novel L particles were not merely virions which had formed without the inclusion of a nucleocapsid or virions which had subsequently lost their nucleocapsid during preparative handling. Thus these novel L particles are genuine products of the infectious processes occurring when HSV-1 replicates.

The nonstructural virus-specific NS1 protein of tick-borne encephalitis (TBE) virus was found to be the main antigenic component of the nonvirion ("soluble") antigen which had been shown previously to present a complex protein structure closely associated with the infected cell membranes. In the presence of sodium dodecylsulphate, beta-mercaptoethanol and 8M urea NS1 protein could be detected in the form of oligomeric molecules which disintegrated to monomers after heating at 100 degrees C for 5 min. The infected cell membrane-associated NS1 protein was shown to differ in electrophoretic mobility from the NS1 protein released into the culture fluid in the course of virus infection. The conditions for detection of NS1 protein and its oligomeric forms by PAG electrophoresis and immune blotting were determined. Dimeric forms of NS1 protein were found to contain products of its proteolysis. NS5 protein was found together with NS1 protein in TBE nonvirion antigen and also possessed antigenic activity.

The rotational mobility of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase) was determined by using erythrosin (ER)-labeled monovalent Fab' antibody fragments directed specifically against either F or HN. By use of time-resolved phosphorescence anisotropy, the rotational mobility of Er-Fab'-viral glycoprotein complexes was studied both in the envelopes of unfused virions bound to erythrocyte ghosts and in the target cell membrane after fusion had occurred. The rotational correlation times (phi) of Er-Fab'-labeled F and HN were rather similar in the envelopes of bound unfused virions, but highly different in membranes of fused cells. The different phi values indicate that F and HN diffuse separately in the target cell membrane and for the major part are not complexed together. The temperature dependence of the phi values of the Er-Fab'-viral glycoprotein complexes revealed a breakpoint at 22 degrees C for the F protein both in bound virions and in the membranes of fused cells, and for the HN proteins in the envelopes of bound virions. In all these cases, the phi values increased between 4 and 22 degrees C, demonstrating a reduction in the rate of rotational diffusion. Further elevation of the temperature reversed the direction of the change in phi. This phenomenon may reflect a temperature-dependent microaggregation of F and HN saturating at ca. 22 degrees C and presumably related to the fusion mechanism since the breakpoint temperature correlates closely with the threshold temperature for virus-cell and cell-cell fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

In the culture fluid from cells infected with feline calicivirus (FCV) F4 strain, the infectious and smaller non-infectious subunit particles were detected by complement fixation (CF) test after sucrose gradient centrifugation. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses confirmed the existence of the subunit particles of FCV, and showed that the infectious and subunit particles were mainly composed of 65K capsid protein. The subunit particles were further purified by ion-exchange chromatography and sucrose gradient centrifugation. The purified subunit and infectious particles had the same neutralizing epitope on 65K protein detected by immunoblot analysis with a neutralizing monoclonal antibody. Antigenic comparison between the infectious and subunit particles by the CF tests using an antiserum against heterologous strain of FCV F14 indicated that the subunit particles might have more highly conserved antigens of FCV than the infectious particles.

The role of zinc in retroviral gag protein function has been addressed through the application of high-resolution nuclear magnetic resonance spectroscopy to samples of the nucleocapsid protein (NCP, p7) isolated directly from infectious HIV-1 particles. Unlike reports for the NCP from avian myeloblastosis virus (AMV) particles [Jentoft et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7094], we find that the HIV-1 NCP binds 2 equiv of zinc tightly and stoichiometrically. Two-dimensional NMR spectroscopic studies reveal that zinc binding induces formation of folded domains that are conformationally similar to (if not identical with) structures observed previously for relevant retroviral-type (RT) zinc finger peptides [formerly called zinc fingerlike peptides; Summers et al. (1990) Biochemistry 29, 329]. This finding is consistent with the hypothesis that the inability of mutant proteins (with substituted Cys and His residues) to package viral RNA results from deficient zinc-binding capability, which may have significant consequences in the development of vaccines for the prevention of AIDS.

Recently D.D. Dunigan, R.G. Dietzgen, J.E. Schoelz, and M. Zaitlin (Virology 165, 310-312, 1988) demonstrated that a small proportion of the subunits of tobacco mosaic virus particles were conjugated with the small protein ubiquitin. We have now detected ubiquitinated conjugates in immunoblots of virion preparations of several other plant viruses, using anti-human ubiquitin antiserum. Based on their polyacrylamide gel migrations, plant virus-associated ubiquitin-immunoreactive proteins were considered to be possible virus structural protein-ubiquitin conjugates of the following viruses: barley stripe mosaic, brome mosaic, cowpea mosaic (two proteins), cowpea severe mosaic (two proteins), and satellite panicum mosaic. Ubiquitinated conjugates were not detected in immunoblots of preparations of cucumber mosaic virus and Cymbidium mosaic virus. The significance of ubiquitinated viral proteins remains to be determined.

Monoclonal antibodies (MAbs) directed against the E2 glycoprotein of mouse hepatitis virus (MHV) have been classified according to their ability to bind to either of the two purified 90,000-molecular-weight subunits (90K subunits) of the 180K peplomeric glycoprotein E2. Correlation with previously reported information about these MAbs suggest that both of the subunits of E2 are important for viral infectivity and cell fusion. Incubation of trypsin-treated virions at pH 8.0 and 37 degrees C released only the E2N subunit from virions. The pattern of MAb reactions suggested that a conformational change occurred in the E2N subunit in association with its release from virions under mildly alkaline conditions at 37 degrees C, the same conditions which are optimal for coronavirus-induced cell fusion.

We have identified unconjugated ubiquitin as a component of avian leukosis virus (ALV). Quantitation both by immunoblotting and by protein staining showed that ubiquitin makes up about 0.5% of total viral protein, corresponding to 100 molecules per virion. This level is about fivefold higher than the level of unconjugated ubiquitin in the cytosol, when expressed as a fraction of total protein. Other abundant low molecular weight proteins in the cytosol were not detected in virions, indicating that packaging occurs in a specific manner. A naturally occurring ALV mutant that lacks the env gene was found to package normal levels of ubiquitin, ruling out involvement of the viral glycoproteins as carriers of ubiquitin. We examined disrupted virus particles as well as lysates of infected cells for the presence of gag protein-ubiquitin conjugates. No conjugates could be detected, suggesting that ubiquitin does not enter virions linked to gag protein.

The expression of the preS1 antigen of hepatitis B virus in sera from chronic HBsAg carriers was studied using a specific monoclonal antibody F35.25 in an original, double-immunoradiometric assay. The antibody F35.25 recognized an epitope located between amino-acid residues 32 and 53 on the preS1 sequence of the large HBsAg protein. This domain could be involved in the recognition of hepatitis B virus by hepatocyte receptors. PreS1 antigen detection by monoclonal antibody F35.25 closely correlated with the presence of complete virions in the serum of HBsAg carriers, as demonstrated by ultracentrifugation-gradient experiments and electron-microscopical examination. Of the 19 HBsAg carriers with chronic liver disease, preS1 antigen was detected in 17 (90%): all of the 11 HBeAg- and hepatitis B virus-DNA--positive cases (group 1) and six of eight anti-HBe--positive cases with low levels of hepatitis B virus replication (group 2). PreS1 antigen/HBsAg ratios parallel to preS1 antigen titers were significantly higher in the HBeAg-positive group (34% and 1:10(6] than in the anti-HBe--positive group (18% and 1:10(2]. In contrast, preS1 antigen was not detected in 18 (90%) of the 20 HBsAg healthy carriers positive for anti-HBe and negative for serum hepatitis B virus-DNA (group 3). Our results show that in chronic HBsAg carriers the serum expression of preS1 antigen correlates well with the level of hepatitis B virus replication (serum hepatitis B virus-DNA and/or liver HBcAg) and that it may be useful in assessing the clinical importance of the chronic viral infection.

Treatment of virions of human immunodeficiency virus type 1 (HIV-1) with ionic and nonionic detergents (NP-40, octylglucoside, sodium deoxycholate) exerted an effect on the virus uncommon for enveloped viruses: instead of solubilization, both glycoproteins (gp120 and gp41) were found in subviral particles, whereas the core protein p24 was found in the supernatant fluid after the removal of subviral particles by centrifugation. The matrix protein p17 and unprocessed molecules of the precursor protein p55 were associated with subviral particles. The above data confirm the proposed model of the HIV-I structural organization according to which glycoproteins are incorporated into the isometric matrix formed by protein p17. Our data indicate that the core protein p24 is not incorporated into the matrix and not associated with nucleocapsid proteins.

Enveloped viruses, of which the orthomyxo- and paramyxoviruses are members, are known to be uncoated by nonionic detergents in a salt concentration-dependent manner. In this study we have shown that detergent uncoating of myxoviruses depends not only on salt concentration but also on pH. Treatment of orthomyxoviruses with Nonidet-P40 or Triton N-101 at low salt concentrations results in solubilization of surface virion glycopolypeptides in alkaline and neutral pH (9.0-6.5), but in acidic pH (6.0-5.0) the viral matrix protein M1 is also removed, and the viral ribonucleoprotein complex is released. Conversely, the paramyxovirus matrix protein M is more completely solubilized in alkaline pH (pH 9.0) than in neutral and acidic pH 7.4-5.0. The described pH-dependent differences are discussed in terms of orthomyxo- and paramyxovirus uncoating in target cells.

Inovirus (filamentous bacteriophage) is a simple system for studying the rules by which protein primary structure (amino acid sequence) controls secondary and higher order structure, and thereby function. The virus occurs naturally as a number of different strains with similar secondary and higher order structure, but the protein subunit that assembles to form the virion coat has quite different primary structures in different virus strains. Despite these differences in primary structure, the subunits of all strains have much the same size, about 50 residues, which are distributed by type in much the same way into three domains of primary structure: a collection of acidic residues in the N-terminal region, a hydrophobic domain of about 19 residues near the middle, and a collection of basic residues near the C-terminus. Each subunit can be closely approximated by an alpha-helix with its long axis roughly parallel to the fibre axis, sloping from large to small radius in the virion and interleaving between subunits in the next turn or level. The acidic residues near the N-terminus of the subunit face outwards on the virion surface, and explain the low isoelectric point of the virion; the basic residues near the C-terminus face inwards, where they neutralize the charge on the DNA at the core of the virion; and the hydrophobic central domain is involved in interactions which bind neighbouring subunits. Detailed X-ray fibre diffraction analysis of one strain gives the subunit structure. Comparative model-building studies of different strains illustrate the common structural principles.

The influence of the conditions of adsorption and virion destruction by freezing-thawing and detergents on the detection of M1 and NP proteins of different influenza virus strains by solid-phase enzyme immunoassay with direct virion adsorption on polystyrene was studied. It was found that for the detection of M1 protein the optimal conditions included virion disruption with detergent and adsorption to polystyrene at 4 degrees C, and for NP protein disruption by freezing-thawing at adsorption to polystyrene at 37 degrees C. In the study of the antigenic properties of protein M1 of different influenza virus strains using monoclonal antibodies it was shown to be necessary, first, to achieve maximum detection of proteins and, second, to standardize the amount of the adsorbed antigen with polyclonal antibodies.

The virus capsomeres of the outer and inner layers of capsids were effectively released simultaneously from purified virions by lipase digestion and were purified by a linear gradient ultracentrifugation. The capsid consisted of an array of double layers of uniformly arranged individual capsomeres where a lipid(s) served as a matrix in between the capsomeres.

A virus-like particle (VLP) of 35 nm diameter has been isolated from the lipolytic yeast Candida curvata. The VLP contains a linear, double stranded RNA molecule of 1.55 microns in length.

Some physico-chemical and molecular-biological parameters of B-lymphotropic herpes virus of Macaca arctoides (HVMA) have been analyzed. The buoyant density of virions and nucleocapsids in a gradient of the percoll density is 1.10 and 1.14 g/ml, respectively, while that of DNA in CsCl gradient is 1.717 g/ml, which is typical for herpes viruses of primates. At the same time the homology of DNA HVMA with DNA EBV and DNA HVP is 30-45%. Differences in restrictions sites between DNA HVMA, DNA HVP and DNA EBV are determined. The intraspecific heterogeneity of HVMA strains has been detected according to restriction sites of DNA. The HVMA genome size is about 170 kb. The DNA HVMA is colinear to the EBV genome, which is also typical of B-lymphotropic herpes viruses of primates.

While studying the haemagglutinin content of whole virus inactivated influenza vaccines by the single radial diffusion test and quantitative electron microscopy, it was found that not all haemagglutinin measured by single radial diffusion was bound to virions, a part of it being in a free state. The influence of unbound haemagglutinin on the immunogenicity of whole virus inactivated influenza vaccine is discussed. In addition, the use of single radial diffusion for the assessment of unbound haemagglutinin is suggested.

The distribution of DNA topoisomers in intracellular simian virus 40 DNA was analyzed by gel electrophoresis. The results suggested that DNA extracted from 70S chromatin had a different superhelical density distribution as compared with the DNA obtained from virions or virion assembly intermediates. The heterogeneity of simian virus 40 viral DNA superhelical density at a late time after infection was partly due to increased virion production and partly due to the intrinsic heterogeneity of the superhelical density of DNA extracted from virions. Using two-dimensional gel electrophoretic analysis we also showed that simian virus 40 DNA templates used for DNA replication have a higher average superhelical density than the bulk of intracellular viral DNA.

Feline Viral Rhinotracheitis herpesvirus (Feline Herpesvirus 1; FHV-1) was purified by rate zonal isopycnic centrifugation. Viral nucleocapsids were isolated from the nuclei and purified. Analysis of the purified and radiolabelled viral polypeptides and glycoproteins by gradient SDS polyacrylamide gel electrophoresis revealed that 23 viral proteins and 6 glycoproteins were present in the whole virus particle. Tween 80-diethylether treatment removed the major glycoproteins. FHV-1 viral glycoproteins were detected using [14C] glucosamine and a lectin binding procedure. The fluorescein isothiocyanate-Concanavalin A (FITC-Con A) specificity was checked by trials on other viruses whose glycoproteins had already been described. The FITC-Con A allows a very simplified procedure for glycoprotein determination after slab acrylamide gel electrophoresis and can be used prior to Coomassie blue staining of proteins.

Two main modifications of the simian virus 40 chromatin were found to occur during the lytic cycle. One was the progressive increase in the acetylation level in the four non-H1 histones as the 75S deoxynucleoprotein complexes (minichromosomes) became assembled into heavier structures. The other was the final elimination from viral chromatin of histone H1. An important stage in the course of these changes was represented by an intracellular simian virus 40 particle, in which the virus-coded proteins were already assembled, but properties distinct from those of mature virions were still present. This particle resembled the mature virions in morphology, sedimentation rate, and buoyant density. It was distinguished by the instability, the presence of histone H1, the uptake of radioactive acetate, and the lower infectivity. Its significance appears to be that of an immature virion on the basis of these characters and of the consistent kinetic behavior during the lytic cycle.

Theiler's murine encephalomyelitis viruses (TMEV) are separable into two groups based on their biological behavior: those highly virulent isolates which are unable to cause persistent infection and the less virulent isolates which regularly produce persistent central nervous system infection in mice. Two highly virulent and five less virulent TMEV were found to have the same buoyant density (1.34 g/ml) on isopycnic centrifugation and virion structure by electron microscopy. Negatively stained virus particles purified in Cs(2)SO(4) gradients appeared to have icosahedral symmetry and measured 28 nm in diameter. Mature virions were found to possess three major structural polypeptides, VP1, VP2 and VP3, in the range of 25,000 to 35,000 daltons, and a smaller fourth major polypeptide, VP4, of 6,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The precursor of VP2 and VP4, VP0, which is a minor polypeptide of mature picornavirus particles, was also identified. However, a slight but consistent difference in several of the capsid polypeptides between the highly virulent and less virulent TMEV was found. VP1 was slightly larger (34,000 versus 33,500 daltons) and VP2 was slightly smaller (31,000 versus 32,000 daltons) for the highly virulent strains compared to the same polypeptide species in the less virulent viruses. VP0 was also slightly smaller (35,500 versus 36,000 daltons) for the highly virulent isolates compared to their less virulent counterparts. Finally, trypsin which was used initially in our purification procedure resulted in preferential cleavage of a 2,000-molecular-weight fragment or fragments from VP1 of only the less virulent isolates.

A new protein has been detected in the nuclei of vaccinia virus-infected cells. This protein has an apparent mol. wt. of 28000 (VP28) on SDS--polyacrylamide gels and has been detected in Triton X-100-treated nuclei of infected BSC-40, L-929 and CVC cells. Within the infected cells, VP 28 was synthesized maximally at 1 to 2 h p.i. in the cytoplasm and accumulated in the nuclei at 4 to 5 h p.i. The appearance of VP28 was not affected by cytosine arabinoside (25 microgram/ml), an inhibitor of virus DNA synthesis, or rifampicin (100 microgram/ml), an inhibitor of vaccinia assembly, but was inhibited by irradiation of the infecting virions; thus classifying it as an early vaccinia virus gene product. Nuclear--cytoplasmic mixing experiments suggested that the nuclear location of VP28 was not an artefact of the cell fractionation techniques employed. VP28 did not appear to be phosphorylated.

A comparison was made between two alternative methods for the nuclear extraction of Simian virus 40 (SV40) virions and nucleoprotein complexes (NPCs) from SV40-infected TC7 cells. The low-salt hypotonic method of Su and DePamphilis (1976) was compared with the detergent method of Garber et al. (1978), since other methods had been shown to result in virion breakdown. There was no disruption of mature SV40 virions with either of these extraction procedures. There was, however, considerably more effective extraction of SV40 NPCs, known to contain large tumor (T) antigen, using the low-salt hypotonic method as opposed to the detergent method. Thus, the low-salt hypotonic method for extraction should be the method of choice when studying SV40 DNA replication or the function of SV40 T antigen in SV40 nucleoprotein complexes.

Trypsin- and acetone-treated virions from either of two strains of measles virus grown in Vero cells stimulated the production in guinea pigs of (i) virus-specific antibodies to polypeptide (P) of molecular weight 70,000 (70K) and to the portion of the HA spike embedded in the viral envelope, but not to M protein, and (ii) antibodies to two host cell membrane antigens which were identified as glycoproteins with molecular weights of 108K and 104K. These host cell antigens were present in increased amounts in infected cells and were intimately associated with the virus. Untreated measles virions grown in Vero cells also stimulated the production of antibody to the 108K glycoprotein. The host polypeptides were less antigenic in virus derived from HEp2 cells, which apparently contained less of these antigens.

A new method for the isolation of simian virus 40 nucleoprotein complexes from nuclei of lytically infected cells is described. The method is based on the addition of a thiol-specific reagent, 5'5'-dithiobis(2-nitrobenzoic acid), to lysis and extraction buffers. By inhibiting an uncoating activity during simian virus 40 extraction, 5'5'-dithiobis (2-nitrobenzoic acid) allows the use of efficient extraction buffers, such as one containing Triton X-100 and EDTA, for the isolation of native simian virus 40 minichromosomes and virion-type structures. Use of the method is illustrated by following encapsidation of simian virus 40 minichromosomes in a pulse-chase experiment. Since 5'5'-dithiobis (2-nitrobenzoic acid) is an inhibitor of many different enzymes, the 5',5'-dithiobis (2-nitrobenzoic acid) extraction technique may be useful for the isolation of not only papovaviruses but also other viruses and possibly cellular chromatin.

Herpesvirus saimiri particles were purified from productively infected owl monkey kidney cell cultures, and the virion polypeptides were analyzed by polyacrylamide gel electrophoresis. A total of 21 predominant proteins were found in lysates of H. saimiri 11 particles by Coomassie blue staining or by [35S]methionine labeling and autoradiography; all proteins were between 160,000 and 12,000 daltons in size. They are most probably virion constituents, as most of them were precipitated by immune sera, and no dominant proteins of equivalent sizes were found in mock-infected cultures. Four glycoproteins (gp 155/160, gp 128, gp 84/90, gp 55) and three polypeptides that appeared not to be glycosylated (p71, p35, p28) were assigned to the envelope or matrix of virions, whereas at least four phosphoproteins (pp132, pp118, pp55, pp13) and ten polypeptides without apparent secondary modification (p155/160, p106, p96, p67, p53, p36, p32, p15, p14, p12) were found in the nucleocapsid fraction. Analysis of virion proteins from different H. saimiri strains did not reveal appreciable differences in the migration behavior of most polypeptides, including all glycoproteins; however, determination of a strain-specific size pattern was possible for three of four phosphoproteins. The overall similarity in protein architecture of H. saimiri strains obviously does not reflect the variability in biology, such as oncogenic properties. In comparison, DNA sequence divergences appear to remain a better taxonomic criterion for strain distinction.

Phenotypic transformation of the host cell affected the formation of polyoma pseuodovirions. Polyoma virus infection of various transformed derivatives of mouse 3T3 cells resulted in the formation of predominantly pseudovirions, whereas infection of mouse 3T3 cells produced mainly polyoma virus. The effect that transformation of the host cell had on polyoma pseudovirus formation was further demonstrated by using phenotypic revertants isolated from some of the transformed cell lines. The revertants were characterized by their morphology, saturation densities, and colony-forming ability in methylcellulose suspension. By these criteria they were distinct from their transformed parents and similar to 3T3 cells. After infection, the revertants produced predominantly polyoma virus and few pseudovirus. Thus, for the cell lines used in this study, phenotypic transformation enhanced the formationof polyoma pseudovirions.

Seven African swine fever virus structural proteins were localized in the virion by immunoelectron microscopy. African swine fever virus-infected cells were incubated, before or after embedding and thin sectioning, with monoclonal antibodies specific for different structural proteins, and after labeling with protein A-gold complexes, the samples were examined in the electron microscope. Proteins p14 and p24 were found in the external region of the virion, proteins p12, p72, p17, and p37 were found in the intermediate layers, and protein p150 was found in the nucleoid and in one vertex. A monoclonal antibody that recognized protein p150 as well as p220, a virus-induced, nonstructural protein, could also bind to a component present in the nucleus of both uninfected and virus-infected cells.

The structural proteins of Western equine encephalitis virus, a member of the alphavirus group, have been characterized by the determination of their amino acid compositions and by N-terminal sequence analysis. More than 60 residues of the N-terminal sequences of each of the envelope glycoproteins have been determined. A comparison of these sequences with the previously determined sequences of two related alphaviruses. Sindbis virus and Semliki Forest virus, strongly supports the view that all three viruses have evolved from a common ancestor and provides information on the pattern of this evolution. The analysis of the capsid proteins of Western equine encephalitis virus shows that the nucleocapsid of this virus can accommodate a considerable degree of variability in its protein component and that at least some regions of alphavirus capsid proteins show more extensive differences between different viruses than do the envelope glycoproteins.

Coxsackievirus A16 (CA16) and enterovirus 71 (E71), which cause hand, foot and mouth disease, were compared with respect to their polypeptide composition by PAGE. Only three proteins were resolved for CA16, whereas the four characteristic structural proteins of E71, (VP1, VP2, VP3 and VP4) were separated. The distribution of labeled proteins suggested that the molecular weights of VP2 and VP3 of CA16 were very similar. The smallest protein, VP4, of CA16 and E71 had the same molecular weight, but the other proteins of the two serotypes were different in their molecular weights.

Dense particles (density 1.44 g/ml) were isolated during purification in CsCl of echovirus 11 produced in HeLa cells. The dense particles had similar antigenic properties and similar RNA and protein composition to standard (density 1.33 g/ml) echovirus 11 particles. They differed from standard particles in their higher buoyant density, lower infectivity and slightly smaller diameter. In contrast to other picornavirus dense particles, echovirus 11 dense particles were present as a major component of virus population. This high ratio of dense particles was found only in virus from HeLa cells which produced non-haemagglutinating echovirus 11. Haemagglutinating echovirus 11 from primary monkey kidney (MK) cells did not contain any detectable levels of dense particles.

Polyoma virus was dissociated into capsomeres (18, 12, and 5S) and a DNA-protein complex (48S) with the Ca2+ chelator, ethyleneglycol-bis-N,N'-tetraacetic acid, and the reducing agent, 2-mercaptoethanol. The reaction was maintained at pH 5.0. Reassembly of the dissociated components to complete virions was accomplished by dialyzing these components overnight at 4 degrees C against the reassembly buffer containing CaCl2, dimethylsulfoxide, Triton X-100, and 0.01 M Tris-acetic acid (pH 5.0). Reconstructed particles ranged from 240S complete virions to lighter intermediate species. Approximately 25% of the dissociated particles could be physically reassembled to complete virions. These virions regained 12.5% of their hemagglutination ability and as much as 6.7% of their original infectivity. The infectivity of these reassembled particles represented a 100-fold increase in infectivity compared with that of the particles that were dissociated and reassembled at pH 7.4. Biochemical analysis showed that the polyoma viral receptor of the virions reassembled at pH 7.4 was greatly reduced, whereas virions reassembled at pH 5.0 retained their receptor. Reassembly could be further improved by additions of either exogenous capsomeres or DNA-protein complex to the reassembly reaction mixture.